Antennae of psychodid and sphaerocerid flies respond to a high variety of floral scent compounds of deceptive Arum maculatum L.

Insect-pollinated plants often release complex mixtures of floral scents to attract their pollinators. Yet scent compounds eliciting physiological or behavioural responses in pollinators have only been identified in few plant species. The sapromyiophilous aroid Arum maculatum releases a highly diverse dung-like scent with overall more than 300 different compounds recorded so far to attract its psychodid and other fly pollinators. The volatiles’ role in pollinator attraction is mostly unknown. To identify potential behaviourally active compounds, we recorded electroantennographic responses of four Psychodidae and one Sphaeroceridae species to (1) inflorescence scents of A. maculatum and (2) the scents released by cow dung, likely imitated by the plant species. Here we show that these flies are sensitive to 78 floral volatiles of various chemical classes, 18 of which were also found in cow dung. Our study, which for the first time determined physiologically active compounds in the antennae of Psychoda spp. and Sphaeroceridae, identified various volatiles not known to be biologically active in any floral visitors so far. The obtained results help deciphering the chemical basis that enables A. maculatum and other plants, pollinated by psychodids and sphaerocerids, to attract and deceive their pollinators.


Scientific Reports
| (2022) 12:5086 | https://doi.org/10.1038/s41598-022-08196-y www.nature.com/scientificreports/ the plant-pollinator interactions 47,48 . Here, we investigated the antennal responses of four psychodid and one sphaerocerid species to the inflorescence scents of A. maculatum and to scent released by cow dung. Specifically, we asked (1) which of the many compounds recorded from this plant species elicit antennal responses in inflorescence visitors, (2) whether antennal responses differ among insect species, and between sexes within species, and (3) how many of the EAD-active scent compounds are shared between A. maculatum and cow dung. The study overall aimed to identify potential scent candidates responsible for attracting and deceiving different pollinators of A. maculatum.
In 2017 and 2018, containers (5 L microboxes, Model: TP5000 + TPD5000-18.5 cm × 18.5 cm × 19.1 cm; Combiness nv, Nevele, Belgium) filled with fresh cow dung (c. 2 L) were offered to insects for three consecutive days as oviposition substrate at the Salzburg site (April-October). Afterwards, the containers were brought to the lab. Once psychodids and sphaerocerids started to hatch in the boxes, the flies were transferred to a small outdoor flight cage (60 cm × 60 cm × 60 cm; BugDorm, Talchung, Taiwan), in which they were offered different breeding substrates (soil, leaf litter, mushrooms, cow or horse dung). Insects reproduced in the cage and were available for electrophysiological measurements for four to six weeks, depending on the species. In spring 2019 and 2020, all insects used for measurements were obtained by bagging A. maculatum individuals with mesh bags at both sites in the morning of the second day of anthesis, prior to the release of trapped insect visitors. Once released by the plant and trapped in the bag, the flies were transferred to a flight cage and bred as described above.

Floral scent collection, electrophysiological analyses (GC-EAD), and identification of EAD-active compounds.
To acquire solvent scent samples for electroantennographic analyses, we collected inflorescence scent of A. maculatum and volatiles released by cow dung using dynamic headspace methods, following 44,49 . Plant volatile samples were collected from a total of eight populations (see Supplementary information Table S1), covering most of the observed scent diversity of A. maculatum 5 , whereas dung volatiles were obtained from cow dung samples (fresh or 1-day old) used for the rearing of flies (see above). Each inflorescence was enclosed in a plastic oven bag (c. 30 cm × 12 cm; Toppits, Melitta, Germany) on the first day of anthesis between 17:30 and 20:00, when scent emission is strongest 5,22 . Circa 60 mL dung was placed into 250 mL glass jars, covered with a plastic oven bag (see above). Volatiles were collected on adsorbent tubes (length: 8 cm, diameter: 2 mm), filled with a mixture of Tenax-TA (mesh 60-80) and Carbotrap B (mesh 20-40; 10 mg each; both Supelco, Germany), that were inserted through small holes into the headspace of the inflorescence and dung each. Samples were collected for 0.5-1.5 h with a flow of 100 mL min −1 , generated by a battery-operated vacuum pump (rotary vane pump G12/01 EB, Gardner Denver Austria GmbH, Vienna, Austria). Due to the thermogenic activity of the inflorescence 22,50 , we partly opened the plastic oven bag at the top to avoid strong condensation of water inside the bag. Samples were eluted from each adsorbent tube using 80-100 µL acetone (SupraSolv, Merck KgaA, Germany; following 49,51 ). Then, samples were pooled per population (plant) or per age (dung, i.e., fresh or 1-day-old) to be used for the physiological measurements. To confirm physiological responses to specific compounds, we recorded antennal responses to mixtures of synthetic compounds for a subset of compounds found in A. maculatum (Table 1, Supplementary information Table S1). Electrophysiological measurements were performed with a gas chromatograph (Agilent 7890A, Santa Clara, California, USA) equipped with a flame ionization detector (FID) and an electroantennographic detection system (GC-EAD) 44,49 . One microliter of a solvent scent sample was injected in splitless mode (250 °C), with hydrogen as the carrier gas (column flow: 3 mL min −1 ). During the period of testing (2017-2020), the GC was equipped with three different columns. In 2017, it was a ZB-5 fused silica column (5% phenyl polysiloxane; 30 × 0.32 mm, 0.25 µm film thickness; Phenomenex, Torrance, CA, USA), which was replaced in 2018 by a chiral fused silica capillary (30 m × 0.23 mm I.D.), coated with a 0.23 μm film of 0.4% heptakis (2,3-di-O-methyl-6-O-tertbutyldimethylsilyl)-β-cyclodextrin (DIME-β-CD) (30%) in SE-52 (70%), the same as described in 52 and 53 . From mid-2019 onwards, the GC was equipped with another DIME-β-CD chiral column (MEGA-DEX DMT Beta SE, 30 m × 0.25 mm ID, 0.23 µm film thickness, MEGA S.r.l., Legnano, Italy). The end of each column was split into two capillaries by a μFlow splitter (Gerstel, Mühlheim, Germany), with nitrogen (N 2 ) as make-up gas (flow rate 25 mL min −1 ). One of the capillaries (2 m × 0.15 µm inner diameter) led to the FID and the other (1 m × 0.2 µm inner diameter) to the EAD setup. The EAD was set up by a transfer line, heated at 220 °C, and a 2-channel USB acquisition controller (Syntech, Kirchzarten, Germany). The outlet of the EAD was placed in a cleaned, humidified airflow, directed onto the mounted antenna. Prior to measurements, each fly was anaesthetised with CO 2 , and the head and last antennomere (apical-tip) were removed. Subsequently, the head and one randomly selected antenna were each connected to a glass micropipette electrode, filled with 95% insect Ringer's solution (8.0 g L −1 NaCl, 0.4 g L −1 KCl, 4.0 g L −1 CaCl 2 ) and 5% Tween 20 (Sigma Aldrich, Vienna, Austria), andconnected to silver wires. The recording electrode was attached to the tip of the antenna, while the reference electrode was connected to the caudal side of the head 44,54 .
Solvent scent samples of A. maculatum were tested on the antennae of five Diptera species: the Sphaeroceridae Coproica ferruginata stenh. (one female) and four Psychodidae species, i.e., Psychoda phalaenoides (12 females and eight males), P. zetterstedti (two females and two males), P. trinodulosa (one male), and P. cinerea banks (two females) ( Table 1, Supplementary information Table S1). All these fly species are visitors of A. maculatum 24,25;27 , except for P. cinerea, which is a pollinator of A. hygrophilum and A. italicum 29 , with the latter sharing several floral compounds with A. maculatum 21,32 . Two additional female Psychoda individuals (collected directly from and Coproica ferruginata (Sphaeroceridae). Compounds are sorted according to chemical classes, and within those alphabetically. The volatiles printed in bold were also detected in cow dung. In parentheses, the numbers of individuals tested (n) and runs performed per fly species and sex are denoted. Superscript values indicate the amounts (ng per injected sample; mean±SD) of volatiles tested in the GC-EAD measurements on different fly species and sexes. Levels of EAD-activity: + + + + (EAD-active in > 80% of runs with samples that contained this compound), + + + (80-50%), + + (< 50-25%), + (< 25%),-(VOC did not elicit a signal), NP (VOC not present in scent samples tested). * The physiological activity was confirmed by a synthetic standard (see Supplementary information www.nature.com/scientificreports/ A. maculatum) could not be determined to species level, as their abdomens were too damaged, and might belong to the four above mentioned species or (an)other species. For identification of EAD-active compounds, scent samples were run on a gas chromatograph/mass spectrometer (GC/MS, model QP2010 Ultra EI, Shimadzu, Tokyo, Japan), equipped with either a non-chiral ZB-5 column (in 2017; see above) or a chiral column (2018-2020; MEGA-DEX DMT Beta SE, see above). Helium was used as carrier gas (flow: 3 mL min −1 ) and samples (injection volume: 1 µL) were run with a split ratio of 1:1 44,49 . Obtained data were handled using GCMSolution v.4.41 (Shimadzu Corporation, Kyoto, Japan). We tentatively identified components by comparison of Kováts' retention indices 55 (KRIs; based on commercially available n-alkanes C 7 -C 20 ) and mass spectra available in the libraries of Adams 56 , FFNSC 2, Wiley9, NIST11, and ESSENTIAL OILS (available in MassFinder 3, Hochmuth Scientific Consulting, Hamburg, Germany). The identity of some of the components was verified by authentic reference standards, available in the collection of the Plant Ecology Lab of Salzburg University (see Table S1). Compounds were classified as inflorescence-specific or as vegetative compounds, according to Gfrerer et al. 5 . Absolute amounts of compounds tested in the GC/EAD measurements (Table 1) were quantified by injecting known amounts of various aliphatics and terpenoids and the resulting mean peak areas were used for quantification 5 .

Results
Across all tested dipterans, we found a total of 78 volatile organic compounds (VOCs) from A. maculatum (together c. 88% of the relative inflorescence scent emission of this species 5 ) that were electroantennographically active. The majority of these compounds were inflorescence-specific, but five of them were vegetative compounds [i.e., (Z)-3-hexenyl acetate, nonanal, limonene, linalool, α-pinene; Table 1]. Overall, 55 of the EAD-active VOCs could be (tentatively) identified (Table 1). They represented several chemical classes, including monoterpenoids (n = 25 VOCs), aliphatic compounds (15), sesquiterpenoids (eight), irregular terpenoids (two), nitrogen-bearing compounds (three), and aromatic compounds (two). Seven of these VOCs elicited antennal responses in all tested insect species: the monoterpenoid 2,6-dimethylocta-2,6-diene (isomer 2), the aliphatic compound 1-octen-3-ol,  Supplementary Table S1 for detailed population information. All samples shown were run on a chiral fused silica capillary column (30% DIME-β-CD in 70% SE-52, see methods) and measurements lasted either 8 (Coproica ferruginata) or 10 min (Psychoda sp.).  Fig. 2). All other VOCs elicited responses only in a subset of insect species. On average, 38 scent compounds yielded a response per species (and sex), with a minimum of 13 volatiles in male P. trinodulosa (Psychodidae; one individual tested on one scent sample), and a maximum of 60 VOCs in female P. phalaenoides (12 individuals tested on nine scent samples). When considering both sexes of P. phalaenoides (total of 20 individuals tested on 16 scent samples), 61 VOCs were EAD-active, and in both sexes of P. zetterstedti 49 VOCs (four individuals on eight scent samples; Table 1, Supplementary information Table S1). For the female C. ferruginata (Sphaeroceridae; one individual tested on three samples), 36 VOCs resulted in an antennal response. Notably, the aliphatic compound 2-nonanone was perceived by all psychodid species, but did not elicit signals in C. ferruginata, even though this compound was present in all three scent samples tested on the latter species. In contrast, the monoterpene γ-terpinene was EAD-active only in C. ferruginata, but not in the two psychodid species tested on this compound, i.e., P. phalaenoides and P. sp.

Scientific Reports
Some compounds elicited specific responses in certain Psychoda taxa. For instance, the aliphatic compound nonanal induced responses in P. phalaenoides and P. zetterstedti, but not in P. cinerea and P. trinodulosa. The nitrogen-bearing compound skatole was EAD-active in P. phalaenoides and P. cinerea, but not in P. zetterstedti and P. sp. ( Table 1). The sesquiterpene α-humulene resulted in responses in most individuals of P. zetterstedti and in a few individuals of P. phalaenoides, but not in P. cinerea and P. sp. A number of the unknown volatiles (e.g., UNK883, UNK1394, UNK1492) elicited responses in P. phalaenoides and P. zetterstedti, but not in P. cinerea.

Discussion
Our study is the first to identify electroantennographically active compounds in Psychoda spp. (Psychodidae) and a Sphaeroceridae (Coproica ferruginata). It shows that these insect visitors of deceptive Arum maculatum are sensitive to a high number of the plants' inflorescence scent compounds. The EAD-active compounds identified represent various chemical classes, including mono-and sesquiterpenoids, aliphatic, aromatic, nitrogen-bearing, and unknown compounds. Antennal responses differed among insect species and between sexes within species. More than a fifth of the physiologically active scent compounds were also released by cow dung, linking insect breeding/mating sites, floral VOCs of A. maculatum, and its floral visitors.
A few of the compounds recorded as physiologically active in the antennae of Psychoda spp. (1-octen-3-ol, butanoic acid, α-pinene, and α-terpinene) were also found to elicit electrophysiological responses in females of the phlebotomine sandfly Lutzomyia longipalpis (Psychodidae, Diptera) 56,57 , the only other psychodid used so far for physiological measurements in the olfactory circuitry. This sandfly, which was tested on faeces from vertebrates and canid host odours, additionally responded to several other volatiles that do not occur in A. maculatum (including different isomers of monoterpenoids), but we provide the first evidence that psychodids are able to perceive sesquiterpenoids. Physiological measurements on antennae of Sphaeroceridae were not available before our measurements, and thus overall, our study increases the knowledge about the peripheral olfactory circuitry of psychodids and Sphaeroceridae.
Altogether five inflorescence scent compounds of A. maculatum have previously been reported as attractive to psychodids. In detail, indole and p-cresol, together with α-humulene or 2-heptanone, were found to attract female P. phalaenoides 41 and Psychoda spp. 34 . In Kite et al. 34  www.nature.com/scientificreports/ mixture of skatole, indole, and p-cresol, together with VOCs not occurring in A. maculatum (geranyl acetone, dihydro-, and β-ionone), was shown to attract psychodid and sphaerocerid pollinators of Typhonium eliosurum, a dung-mimicking aroid endemic to Australia 28 . In preliminary bioassays in the field, we tested the above five compounds, using the same composition and concentration as released by the inflorescences of A. maculatum. Yet those volatiles did not attract psychodid or sphaerocerid flies. This suggests that other, not yet tested scent compounds (additionally) contribute to pollinator attraction in A. maculatum. Potential candidates are other odours known also from cow or horse dung (e.g., 2,6-dimethylocta-2,6-diene, unknown UNK1415; Table 1) or compounds known from other breeding substrates (e.g., fungi: 3-octanone 60 ). The unknown UNK1415, one of the main scent compounds of A. maculatum 5 , yielded antennal responses in all insect taxa and in nearly all individuals tested in the present study. Interestingly, this unknown volatile is possibly identical to unknown "RI 1531" found in T. eliosurum 28 , as both volatiles have the same mass spectra (Supplementary information Fig. S1).
Our study shows that some of the antennal responses to scent differ among insect species, and some also between males and females within species. This finding is in agreement with results obtained by physiological measurements in other insects 43,63 . Some of the species-level effects described in the present study might have been influenced by sex-specific effects, because for some species (P. cinerea and P. trinodulosa) we only tested males or females. Hence, differences in antennal responses among these species need to be interpreted with caution. Nonetheless, species-and sex-specific differences in the peripheral olfactory circuitry of insects can result in different behaviours 64,65 . Interestingly, antennae of P. cinerea, the only non-pollinating species of A. maculatum (but of other Arum spp.) we tested in this study, did not respond to some abundant compounds emitted by A. maculatum (e.g., UNK1394, UNK1492), which otherwise elicited responses in the other pollinating species tested (e.g., P. phalaenoides and P. zetterstedti). The lack of antennal sensitivity to (some of) those compounds might explain why P. cinerea does not visit A. maculatum, while close relatives including P. phalaenoides and P. zetterstedti are (important) pollinators 24,25 .

Conclusions
Until now, it was not known which (and how many) volatile compounds of the complex floral scent of Arum maculatum can be perceived by its floral visitors. Our study identified 78 physiologically active compounds from hundreds of potentially behaviourally active VOCs, which is still a rather high number the psychodid and sphaerocerid flies are sensitive to. Our results thus provide a basis for future studies that aim to understand the floral volatiles of A. maculatum involved in the chemical attraction and deception of its pollinators, and which VOCs guide the flies to their breeding/mating substrates. Some of the EAD-active VOCs (4-terpinenol, α-terpinene, 2-heptanol, 2-nonanol, UNK1503) have recently been shown to be under phenotypic selection in A. maculatum 5 . These compounds and those EAD-active ones shared with the pollinators' breeding substrates (e.g., UNK1415, 3-octanone) are the most promising candidates for future behavioural assays. As the tested Diptera species (Psychodidae, Sphaeroceridae) are also known pollinators of other (similarly-scented) species of Arum (e.g., A. italicum, A. concinnatum 31,66 ) as well as other species/genera of Araceae (e.g., Typhonium eliosurum 28 ), our study should also help to elucidate the chemical interactions between these plants and their fly pollinators. Future research is now needed to test the behavioural function of physiologically active floral volatiles, which is crucial for a better understanding of olfactory cues mediating plant-animal interactions in general, and in sapromyiophilous species, in particular.

Experimental research and field studies on plants
All samplings were carried out in compliance with the current laws of the respective countries.

Data availability
All data that support the findings of this study are included in this published article (and its supplementary information files).